This invention relates to a method for the isolation of high purity polyhydroxy cyclic carboxylic acids (PCCA), such as shikimic acid, from aqueous solutions. More particularly, this invention relates to the isolation of shikimic acid from a fermentation broth through the use of an organic acid such as acetic acid.
This invention relates to PCCAs and to derivatives of PCCAs that are isolated in high purity from aqueous solutions, such as fermentation broths produced by microorganisms. As used herein, PCCAs are a group of primarily organic molecules that possess two or more hydroxyl groups, a non-aromatic ring structure, at least one carbonyl group and a water solubility of at least 10 gms per 100 ml at room temperature. Representative PCCAs include shikimic acid, quinic acid, 5-dehydroshikimic acid, 6-fluoroshikimic acid and derivatives thereof. A preferred PCCA in the process of the present invention is shikimic acid. Shikimic acid is an intermediate metabolite in the common or shikimate pathway of plants and microorganisms. PCCAs are useful as starting materials for antibacterial, antifungal and herbicidal agents and as nutritionals. The invention in general is directed to novel processes for the preparation and isolation of these compounds.
The shikimic acid pathway is essential for the existence of bacteria and plants as it provides for the synthesis of necessary metabolites. In bacteria, the pathway provides not only the three (3) aromatic xcex1-amino acids (tyrosine, tryptophan and phenylalanine) but also paraaminobenzoic acid, parahydroxybenzoic acid, salicyclic acid and the like. The shikimic acid pathway is well known and well reported in the literature.
Shikimic acid (3,4,5-trihydroxy-1-cyclohexene-1-carboxylic acid) has three (3) chiral centers, which make six (6) different optical isomers possible. A preferred product produced according to the present invention is the natural levorotatory L-shikimic acid of the structural formula: 
One embodiment of the present invention relates to the isolation of shikimic acid from a fermentation broth through a crystallization technique utilizing an organic acid, such as acetic acid.
U.S. Pat. No. 3,546,072 to Araki et al. discloses that 5-dehydroshikimic acid can be prepared by culturing a 5-dehydroshikimic acid producing microorganism of the genus Corynebacterium in a culture medium (fermentation broth), containing a carbon source, a nitrogen source, inorganic material and nutrients. The produced and accumulated 5-dehydroshikimic acid is isolated from the culture medium, after filtration to remove the microbial cells, through an adjustment of pH and the addition of active carbon. The active carbon absorbs the 5-dehydroshikimic acid and is eluded with 98% ethanol. The eluate is concentrated under reduced pressure and then the product precipitated from ethanol. This reference does not suggest or disclose the use of an organic acid such as acetic acid to enhance the precipitation of highly pure shikimic acid crystals from an aqueous fermentation broth.
U.S. Pat. No. 4,769,061 to Comai discloses a genetically modified plant wherein a gene encoding for a mutated glyphosate resistant 5-enolpyruvyl-3-phosphoskimimate synthase enzyme is included in the genome of the plant. This reference makes no suggestion of how to isolate shikimic acid from the tissue of such a genetically modified plant.
U.S. Pat. No. 5,214,165 to Sutherland et al. relates to 6-fluoroshikimic acid derivatives that have antibacterial, antifungal and herbicidal activity. This reference does not suggest the use of genetically modified microorganisms nor the use of glyphosate addition to a fermentation process to increase the production of shikimic acid. This reference further fails to suggest the use of an organic acid to assist in the isolation of shikimic acid from a fermentation broth. This reference does, however, disclose a class of PCCAs that are useful in the process of this invention.
U.S. Pat. No. 5,605,818 to Katsumata et al. discloses a process for producing an aromatic amino acid, such as tryptophan, through the use of culturing in a medium a mutant strain of the genus Corynebacterium or Brevibacterium. These mutant strains are capable of producing the desired aromatic amino acid and also have a higher transketolase activity than that of the parent strain. The desired aromatic amino acid is accumulated in the culture and recovered therefrom. This reference makes no suggestion of the use of an organic acid to isolate the desired PCCA from the broth. This reference does disclose a number of microorganisms that may produce a fermentation broth that is useful in the present invention.
An article entitled xe2x80x9cRecovery of Shikimic Acid Using Temperature-Swing Complexation Extraction and Displacement Back Extractionxe2x80x9d by Miles et al. in Isolation and Purification, 1994, Vol. 2, pp. 75-82, discloses a process for the removal of shikimic acid from aqueous solutions. The Miles et al. process is accomplished through solvent extraction using tridodecylamine dissolved in n-heptanol or n-butanol and back extraction to water using oleic acid to displace the shikimic acid from the organic phase. This reference focuses on developing a general method for recovering metabolic acids from fermentation broths. It fails, however, to disclose the use of acetic acid, which is added to a concentrated fermentation broth, to enhance the precipitation of shikimic acid from the broth.
An article from Synthesis of February, 1993 entitled: xe2x80x9cThe Biosynthesis and Synthesis of Shikimic Acid, Chorismic Acid and Related Compoundsxe2x80x9d, pp. 179-193 by Campbell et al. teaches that compounds other than glyphosate may interfere with the shikimic acid pathway. This article also provides a good description of the glucose derived shikimate pathway and the various arduous approaches to the chemical synthesis of shikimic acid. This article makes no suggestion regarding the isolation of highly pure PCCA from reaction mixtures through the use of concentrated acetic, lactic and/or propionic acids.
In a publication by Jiang, et al. in Tetrahedron Report Number 449, Vol. 54 (1998), pp. 4697-4753, entitled: xe2x80x9cChemical Synthesis of Shikimic Acid and Its Analoguesxe2x80x9d details are disclosed of the complex and arduous task of the synthesis of shikimic acid and its analogues. Jiang, et al., like Campbell, et al, fails to disclose the present invention.
There is disclosed a method for producing high purity (greater than 92% by wt.) crystals of polyhydroxy cyclic carboxylic acids (PCCA) from an aqueous solution which comprises:
a) concentrating the aqueous solution to a concentration of at least 250 gms of PCCA per liter; and
b) combining said concentrated PCCA with at least one acid selected from the group consisting of acetic acid, lactic acid, propionic acid and mixtures thereof; and
c) isolation of said crystal from said slurry.
There is also disclosed a method for producing high purity crystals of polyhydroxy cyclic carboxylic acids (PCCA) from aqueous solutions thereof comprising the steps of:
a) obtaining a solution of PCCA from any living organism capable of producing PCCA from aromatic amino acids, such as a microorganism (such as recombinant E. coli) or a plant. For example, the fermentation broth of a microorganism may be used to form a solution of PCCA;
b) concentrating the solution to a concentration of at least 450 gms of PCCA per liter;
c) combining the concentrated solution with at least one acid selected from the group consisting of acetic acid, lactic acid, propionic acid and mixtures thereof at a temperature of from 25 to 90xc2x0 C. to form a PCCA/acid slurry;
d) cooling the PCCA/acid slurry to a temperature of from 25 to 5xc2x0 C. to form crystals of the PCCA; and
e) isolating the crystals from the slurry.
The PCCAs of the present invention comprise shikimic acid, quinic acid, 6-fluoroshikimic acid, dehydroshikimic acid and derivatives thereof and is, more preferably, shikimic acid.
There is further disclosed a method for producing high purity crystals of shikimic acid, the method comprising the steps of:
a) culturing a microorganism capable of producing aromatic amino acids in a medium by employing means to enhance the excretion of shikimic acid into the medium;
b) separating the microorganism from the medium to obtain an aqueous solution of shikimic acid;
c) concentrating the solution to a concentration of at least 450 gms of shikimic acid per liter to obtain a concentrated solution;
d) combining the concentration solution with concentrated acetic acid at a temperature in excess of 25xc2x0 C. to obtain a shikimic acid/acid slurry;
e) cooling the shikimic acid/acid slurry to a temperature below 25xc2x0 C. to obtain crystals of shikimic acid; and
f) isolating the shikimic acid crystals from the slurry.
In general, the concentrated PCCA aqueous solution is combined with at least one acid selected from the group consisting of acetic acid, lactic acid, propionic acid and mixtures thereof to form a PCCA/acid slurry. This slurry is then cooled to a temperature of less than 25xc2x0 C., preferably less than 10xc2x0 C., most preferably to about 5xc2x0 C., to form high purity crystals. The crystals are then isolated from the slurry using known techniques such as filtration and centrifugation.
There is further disclosed a method wherein the concentration of PCCA is at least 450 grams per liter and which additionally comprises the step of heating and agitating the PCCA/acid slurry to a temperature of from 25 to 90xc2x0 C. prior to the cooling step. The cooling step is preferably accomplished in 1 to 8 hours.
In yet a more preferred embodiment, the concentration of the aqueous solution is at least 500 gms per liter, most preferably at least 600 gms per liter PCCA.